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Novel diversity in Lactifluus section Gerardii from
Asia: five new species with pleurotoid or small
Eske De Crop, Felix Hampe, Komsit Wisitrassameewong, Dirk Stubbe, Jorinde
Nuytinck & Annemieke Verbeken
To cite this article: Eske De Crop, Felix Hampe, Komsit Wisitrassameewong, Dirk Stubbe,
Jorinde Nuytinck & Annemieke Verbeken (2018): Novel diversity in Lactifluus section Gerardii
from Asia: five new species with pleurotoid or small agaricoid basidiocarps, Mycologia, DOI:
To link to this article: https://doi.org/10.1080/00275514.2018.1508979
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Published online: 21 Sep 2018.
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Novel diversity in Lactifluus section Gerardii from Asia: five new species with
pleurotoid or small agaricoid basidiocarps
Eske De Crop
, Felix Hampe
, Komsit Wisitrassameewong
, Dirk Stubbe
, Jorinde Nuytinck
and Annemieke Verbeken
Mycology Research Group, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium;
Institute of Excellence in
Fungal Research, Mae Fah Luang University, 333 Moo 1, Thasud Sub-district, Muang District, Chiang Rai 57100, Thailand;
School of Science,
Mae Fah Luang University, 333 Moo 1, Thasud Sub-district, Muang District, Chiang Rai 57100, Thailand;
Sciensano, Mycology and
Aerobiology Service, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium;
Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The
The ectomycorrhizal milkcap genus Lactifluus (Russulaceae) is commonly found in tropical and
subtropical forests of Southeast Asia. During several sampling expeditions in Thailand, multiple
collections of Lactifluus (Lf.) species with pleurotoid or small agaricoid basidiocarps were found. A
molecular study was combined with a morphological study, in which the collections were
compared with herbarium material. The molecular study indicated that four Thai collections
belonged to undescribed species within Lactifluus section Gerardii, as was also the case for
herbarium collections of a pleurotoid species from Nepal. One other collection from Thailand
appeared closely related to Lf. uyedae, known only from Japan. Five species are described as new
in the genus: Lf. auriculiformis, Lf. gerardiellus, Lf. bhandaryi, Lf. pulchrellus, and Lf. raspei.
Received 19 December 2017
Accepted 3 August 2018
Russulales; taxonomy; 5 new
Lactifluus sect. Gerardii (A.H. Sm. & Hesler) Stubbe was
recently described as a morphologically and molecularly
well-supported section within Lactifluus subg. Lactifluus
(Pers.) Roussel. with a disjunct distribution in North and
Central America and subtropical to tropical Asia and
Australasia (Stubbe et al. 2010,2012a;DeCropetal.
2017). Although often superficially resembling represen-
tatives of Lactarius subg. Plinthogalus (Burl.) Hesler & A.
H. Sm., species of Lactifluus sect. Gerardii can be recog-
nized by a combination of macro- and microscopic char-
acters. In the original description of the section, the
following characteristics were mentioned as most typical:
having a brown stipe and pileus contrasting with the
white, mostly distant lamellae, a white spore print (in
contrast to Lactarius subg. Plinthogalus, where the spore
print is cream to ochraceous), reticulate spore ornamen-
tation not higher than 2 µm (with ridges or intercon-
nected warts), a palisade structure in the pileipellis, and
generally the lack of macrocystidia. Besides these dark-
pigmented and agaricoid representatives (i.e., basidio-
carps characterized by a pileus, lamellae, and a centrally
attached stipe), the group also includes small, white pleur-
otoid species (i.e., basidiocarps characterized by a pileus,
lamellae, and a lateral or absent stipe). As in other clades,
it is now accepted in the Russulales that fruit body shape
has long been overestimated as a phylogenetically con-
served feature, and that agaricoid species are closely
related to angiocarpous and pleurotoid species (De Crop
et al. 2017). Since the recent splitting of milkcaps into
three genera—Multifurca Buyck & V. Hofstetter,
Lactarius Pers. (hereafter abbreviated as L.), and
Lactifluus (hereafter abbreviated as Lf.), it remains a chal-
lenge to find synapomorphic characters for the two large
milkcap genera Lactifluus and Lactarius. The current
morphological delineation is based on several trends
(Verbeken and Nuytinck 2013). One of these concerns
gross fruit body morphology. Milkcaps are mainly agar-
icoid, but angiocarpous and pleurotoid milkcaps do
occur. Angiocarpous species are so far only described in
Lactarius, whereas pleurotoid species are so far only
found in Lactifluus (Verbeken and Nuytinck 2013).
The pleurotoid habit has evolved more than once in
Lactifluus (Stubbe et al. 2010; De Crop et al. 2017), with
eight pleurotoid species in five different clades.
However, all pleurotoid milkcaps are characterized by
a white spore print and thick-walled terminal elements
of the pileipellis. Stubbe et al. (2012b) include three
pleurotoid species in Lf. sect. Gerardii: Lf. conchatulus
CONTACT Eske De Crop firstname.lastname@example.org
Supplemental data for this article can be accessed on the publisher’sWeb site.
© 2018 The Mycological Society of America
Published online 21 Sep 2018
(Stubbe & H.T. Le) Stubbe from Thailand, Lf. uyedae
(Singer) Verbeken from Japan, and Lf. genevievae
(Stubbe & Verbeken) Stubbe from Australia. Latha
et al. (2016) recently described a new Indian pleurotoid
species in the section, Lf. indicus K.N.A. Raj & Manim.
Other pleurotoid species are placed within two other
subgenera of Lactifluus (De Crop et al. 2017). Two
species are known from Lf. subg. Gymnocarpi (R.
Heim ex Verbeken) De Crop: Lf. panuoides (Singer)
De Crop from Central and South America and Lf.
brunellus (S.L. Miller, Aime & T.W. Henkel) De Crop
from Guyana (Miller et al. 2002). Two species are
known from Lf. subg. Lactariopsis (Henn.) Verbeken:
Lf. multiceps (S.L. Miller, Aime & T.W. Henkel) De
Crop from Guyana (Miller et al. 2002) and Lf. chryso-
carpus E.S. Popov & O.V. Morozova from Vietnam
(Morozova et al. 2013). The pleurotoid species
Lactarius campinensis Singer from Brazil was recom-
bined in Russula: R. campinensis (Singer) T.W. Henkel,
Aime & S.L. Mill. (Henkel et al. 2000).
Species with small agaricoid basidiocarps, i.e., with a
pileus diameter mostly less than 2 cm when mature and
with a slender, rather than stout, stature are also
described within different sections of Lactifluus.
Within Lf. sect. Gerardii, the Chinese Lf. parvigerardii
X.H. Wang & Stubbe (pileus 7–15 mm diam; Wang
et al. 2012), the Vietnamese Lf. igniculus O. V.
Morozova & E.S. Popov (pileus 5–16 mm diam;
Morozova et al. 2013) and the Indian Lf. umbonatus
K.P.D. Latha & Manim. (pileus 3–11 mm diam; Latha
et al. 2016) have small agaricoid basidiocarps. Within
Lf. sect. Lactariopsis, one African species is known: Lf.
uapacae (Verbeken & Stubbe) De Crop (pileus
10–14 mm diam; Verbeken et al. 2008); and within Lf.
sect. Edules (Verbeken) Verbeken, also one species is
known from Africa with small basidiocarps: Lf. roseolus
(Verbeken) Verbeken (pileus (5–)10–31 mm diam;
This paper documents three new pleurotoid
Lactifluus species and two species with very small agar-
icoid basidiocarps from south (Nepal) and Southeast
Asia. Furthermore, we document a possible new record
for Lf. uyedae from Thailand.
MATERIALS AND METHODS
Sampling.—We sampled species of Lactifluus subg.
Lactifluus, with a focus on Lf. sect. Gerardii. The
majority of new specimens was collected by the
authors in Chiang Mai, Thailand. Three specimens
were collected in Nepal by Hemanta Ram Bhandary.
We also added 15 representatives of the other Lactifluus
subgenera. For outgroup purposes, we included five
species of the genus Lactarius (TABLE 1). Herbarium
designations follow Thiers (continuously updated).
Morphological analyses.—Macroscopic characters are
based on observation of fresh material. Color codes
refer to Kornerup and Wanscher (1978). Some
macrochemical reactions were also performed on
fresh latex or basidiocarps with 30% KOH and a
tincture of guaiac, respectively.
Microscopic features were studied from dried mate-
rial mainly in Congo red–SDS (sodium dodecyl sul-
fate). Basidiospore ornamentation is described and
illustrated as observed in Melzer’s reagent. For details
on terminology, we refer to Verbeken (1998) and
Verbeken and Walleyn (2010). Line drawings were
made with the aid of a drawing tube at original magni-
fications: 6000× for spores and 1000× for individual
elements and sections. Basidia length excludes sterig-
mata. Measurements are given as min–max, except for
basidiospores. Basidiospores were measured in side
view in Melzer’s reagent, excluding the ornamentation,
and measurements are given as described in Nuytinck
and Verbeken (2005): (MIN)(Ava −2SDa)–Ava–Avb–
(Avb + 2SDb)(MAX), in which Ava/b = lowest/highest
mean value for the measured collections, and SDa/b =
standard deviation of the lowest/greatest mean value.
MIN/MAX = lowest/highest value measured and only
given when they exceed [Ava −2SDa] or [Avb + 2SDb],
respectively. Q stands for “quotient length/width”and
is given as MINQ–Qa–Qb–MAXQ, in which Qa/b =
lowest/highest mean quotient for the measured speci-
mens, and MIN/MAXQ = minimum/maximum value
over the quotients of all available measured spores.
Molecular analyses.—DNA was extracted using the
cetyltrimethylammonium bromide (CTAB) extraction
protocol described in Nuytinck and Verbeken (2003).
Polymerase chain reaction (PCR) amplification
protocols follow Le et al. (2007). We used two nuclear
markers previously shown to be informative within
Lactifluus (Stubbe et al. 2010; De Crop et al. 2017): (i)
nuc rDNA internal transcribed spacer region ITS1-
5.8S-ITS2 (ITS), using primers ITS-1F and ITS4
(White et al. 1990; Gardes and Bruns 1993), and (ii)
nuc rDNA 28S (28S), using primers LR0R and LR5
(Vilgalys and Hester 1990).
PCR products were sequenced using an automated
ABI 3730 XL capillary sequencer (Life Technology at
Macrogen, Amsterdam, The Netherlands). Forward
and reverse sequences were assembled into contigs
and edited where needed in Sequencher 5.0 (Gene
Codes Corporation, Ann Arbor, Michigan). Sequences
2DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
Table 1. Specimens and GenBank accession numbers of DNA sequences used in the molecular analyses.
Species Specimen voucher (herbarium) Country ITS accession no. 28S accession no.
Lf. subg. Lactifluus
Lf. sect. Allardii
Lf. allardii JN 2004-008 (GENT) USA KF220016 KF220125
Lf. sect. Ambicystidiati
Lf. ambicystidiatus HKAS J7008 (HKAS) China KR364108 KR364239
Lf. sect. Gerardii
Lf. atrovelutinus DS 06-003 (GENT) Malaysia GU258231 GU265588
Lf. auriculiformis AV 12-050 (holotype GENT, isotype MFLU) Thailand KR364086 KR364216
Lf. bhandaryi TENN 051830 (holotype TENN) Nepal KR364111 KR364140
Lf. bhandaryi TENN 051831 (TENN) Nepal KX889846 —
Lf. bhandaryi TENN 051832 (TENN) Nepal KX889847 —
Lf. bicolor DS 06-229 (epitype GENT) Malaysia GU258221 GU265577
Lf. conchatulus LTH 457 (holotype GENT) Thailand GU258296 GU265659
Lf. cf. conchatulus EDC 14-502 (GENT) Thailand KX889848 —
Lf. coniculus DS 07-496 (holotype GENT) Sri Lanka GU258236 GU265594
Lf. fuscomarginatus LM 4379 (holotype XAL) Mexico HQ168367 HQ168367
Lf. genevievae GG-DK 17-02-05 (holotype GENT) Australia GU258294 GU265657
Lf. gerardiellus KW386 (holotype GENT, isotype MFLU) Thailand KX889845 KX889844
Lf. gerardii s.l. AV 05-375 (GENT) USA GU258254 GU265616
Lf. gerardii s.l. AV 05-283 (GENT) USA GU258259 GU265621
Lf. aff. gerardii TMI 15534 (TMI) Japan GU258229 GU265586
Lf. aff. gerardii MC 04-259 (GENT) Nepal GU258234 GU265592
Lf. aff. gerardii LTH 394 (GENT) Thailand GU258249 GU265610
Lf. aff. gerardii FRIM 1098 (FRIM) Malaysia GU258232 GU265589
Lf. aff. gerardii DS 07-390 (GENT) Thailand GU258252 GU265613
Lf. aff. gerardii DS 07-373 (GENT) Thailand GU258242 GU265603
Lf. aff. gerardii KIINA 126 (GENT) China GU258227 GU265584
Lf. cf. gerardii var. fagicola JN 2007-029 (GENT) Canada GU258224 GU265582
Lf. hora DS 07-502 (holotype GENT) Sri Lanka GU258238 GU265596
Lf. igniculus LE 262983 (holotype LE) Vietnam JX442759 JX442759
Lf. aff. igniculus LE 253908 (LE) Vietnam JX442760 JX442760
Lf. leae FH 12-013 (GENT) Thailand KF432957 KR364213
Lf. leonardii GG 07-02-04 Australia GU258308 GU265668
Lf. limbatus DS 06-247 (GENT) Malaysia JN388955 JN388987
Lf. cf. ochrogalactus AV-KD-KVP 09-120 (GENT) India KR364130 KR364248
Lf. parvigerardii XHW 2415/KUN F61367 (holotype KUN) China JF975641 JF975642
Lf. petersenii AV 05-300 (GENT) USA GU258281 GU265642
Lf. pulchrellus KW 304/FH 12-037 (holotype GENT, isotype MFLU) Thailand KR364092 KR364223
Lf. raspei EDC 14-517 (holotype GENT, isotype MFLU) Thailand KX889849 —
Lf. reticulatovenosus EH 6472 (holotype GENT) Indonesia GU258286 GU265649
Lf. subgerardii AV 05-269 (GENT) USA GU258263 GU265625
Lf. uyedae MCA 584 (VPI) Japan —AF218562
Lf. cf. uyedae AV 12-070 (GENT) Thailand KR364090 KR364221
Lf. wirrabara s.l. GG 24-01-04 Australia GU258307 GU265667
Lf. wirrabara s.l. PL 40509 New Zealand GU258287 GU265650
Lf. sect. Lactifluus
Lf. acicularis KVP 08-002 (GENT) Thailand HQ318226 HQ318132
Lf. volemus KVP 11-002 (GENT) Belgium JQ753948 KR364175
Lf. sect. Piperati
Lf. piperatus 2001 08 19 68 (GENT) France KF220119 KF241840
Lf. roseophyllus JN 2011-076 (GENT) Vietnam KF220107 KF220202
Lf. sect. Tenuicystidiati
Lf. subpruinosus JN 2011-061 (GENT) Vietnam KR364046 KR364172
Lf. subg. Lactariopsis
Lf. vellereus ATHU-M 8077 (ATHU-M) Greece KR364106 KR364237
Lf. heimii EDC 11-082 (GENT) Tanzania KR364040 KR364167
Lf. cyanovirescens JD 988 (GENT) Congo KR364082 KR364211
Lf. multiceps TH 9154A (BRG, DUKE) Guyana JN168731 —
Lf. chrysocarpus LE 253907 (holotype LE) Vietnam JX442761 JX442761
were aligned using the online version of the multiple
sequence alignment program MAFFT 7 (Katoh and
Toh 2008), using the E-INS-I strategy. We trimmed
trailing ends of the alignment and manually edited
sequences when necessary in MEGA 6 (Tamura et al.
2013). The alignment can be acquired from the first
author and TreeBASE (S19924).
PartitionFinder 1.1.1 (Lanfear et al. 2012) was used
to determine partitions of DNA sequence data. Two
partitions were assigned to partial 18S + 5.8S + partial
28S and ITS1 + ITS2. Maximum likelihood (ML) ana-
lyses were conducted with RAxML 8.0.24 (Stamatakis
2014). One thousand rapid bootstrap replicates were
run under the GTRCAT option (Stamatakis et al.
2008). All analyses were performed on the CIPRES
Science Gateway (Miller et al. 2010).
Our molecular results show that all of the newly collected
species belong to Lactifluus sect. Gerardii (FIG. 1). None of
the new taxa are placed closely to any described or sampled
species, except for Lf. uyedae from Thailand, which is
placed close to the exemplar from Japan. Based on both
molecular and morphological results, we describe five new
species: Lf. auriculiformis,sp.nov.,Lf. pulchrellus,sp.nov.,
Lf. gerardiellus,sp.nov.,Lf. bhandaryi,sp.nov.,andLf.
raspei, sp. nov. Furthermore, we document a possible new
finding of Lf. uyedae in Thailand.
Combining our results with those of previous studies
(Stubbe et al. 2010,2012a;Wangetal.2012), five clades are
recognized within Lf.sect.Gerardii (FIG. 1). (i) The/
gerardii clade contains agaricoid species with large to med-
ium-sized basidiocarps (pileus ca. 1–12 cm diam), with
dark-colored pileus and stipe, reticulate spores, and no
macrocystidia. This clade is weakly supported. (ii) The/
parvigerardii clade contains species characterized by small
agaricoid basidiocarps (pileus ca. 2–16 mm diam) that
display vivid orange-red to brown colors. In addition,
macrocheilocystidia are absent and macropleurocystidia
are present or absent. This clade is strongly supported
(bootstrap [BS]: 100%). (iii) The/fuscomarginatus clade
contains agaricoid species with small to medium-sized
basidiocarps (pileus ca. 1.5–4.6 cm diam) with a dark-
colored pileus and stipe, reticulate spores, and no macro-
cystidia. This clade is strongly supported (BS: 100%). (iv)
The/uyedae clade is highly supported (BS: 93%). Species
from this clade have small pleurotoid or agaricoid basidio-
carps (pileus ca. 2–18 mm diam) with brownish to white
colors, and macrocheilocystidia and macropleurocystidia
are present. (v) The/ochrogalactus clade is also strongly
supported (BS: 100%). Species from this clade have med-
ium-sized agaricoid basidiocarps (pileus ca. 2–8cmdiam)
with latex that changes color after contact with air, pre-
sence or absence of macrocystidia, and spores with rela-
tively high warts connected by fine lines.
Lactifluus auriculiformis Verbeken & F. Hampe, sp.
nov. FIGS. 2A–B,3,4; SUPPLEMENTARY FIG. 1A
Typification: THAILAND. CHIANG MAI PROVINCE:
Doi Suthep-Pui National Park, 18°48.40′N, 98°54.63′E,
Table 1. (Continued).
Species Specimen voucher (herbarium) Country ITS accession no. 28S accession no.
Lf. subg. Pseudogymnocarpi
Lf. aff. rubroviolascens EDC 12-051 (GENT) Cameroon KR364066 KR364195
Lf. luteopus EDC 11-087 (GENT) Tanzania KR364049 KR364176
Lf. rugatus EP 1212/7 (LGAM-AUA) Greece KR364104 KR364235
Lf. xerampelinus MH 201176 (GENT) Mozambique KR364099 KR364231
Lf. armeniacus EDC 14-501 (holotype MFLU, isotype GENT) Thailand KR364127 —
Lf. subg. Gymnocarpi
Lf. gymnocarpus EDC 12-047 (GENT) Cameroon KR364065 KR364194
Lf. panuoides RC/Guy 10-024 (LIP) French Guiana KJ786647 KJ786551
Lf. luteolus AV 05-253 (GENT) USA KR364016 KR364142
Lf. clarkeae MN 2004002 (L) Australia KR364011 HQ318205
Lf. brunellus TH 9130 (BRG, DUKE) Guyana JN168728 —
Lactarius hatsudake FH 12-052 (GENT) Thailand KR364085 KR364215
Lactarius olympianus ED 08-018 (GENT) USA KR364089 KR364220
Lactarius scrobiculatus JN 2001-058 (GENT) Slovakia KF432968 KR364219
Lactarius fuliginosus MTB 97-24 (GENT) Sweden JQ446111 JQ446180
Lactarius tenellus ADK 3598 (GENT) Benin KF133280 KF133313
Note. Accession numbers in bold were newly generated for this study.
4DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
Figure 1. ML tree based on concatenated ITS and 28S sequence data of the genus Lactifluus with a focus on Lf. sect. Gerardii.
Bootstrap values >70% are shown. Red-labeled branch tips are the specimens described in this study. Species with pleurotoid or
small agaricoid basidiocarps are indicated by pictograms in the tree.
Figure 2. Basidiocarps of five Lactifluus species. A. Vertically exposed collecting spot with basidiocarps of Lf. auriculiformis (AV 12-050) in
its natural environment. B. Lactifluus auriculiformis (AV 12-050). C. Lactifluus gerardiellus (KW 386). D. Lactifluus pulchrellus (KW 304/FH
12-037). E. Lactifluus raspei (EDC 14-517), overview ofbasidiocarps.F. Basidiocarp of Lf. raspei under a stereomicroscope. G. Close-up of the
subiculum of Lf. raspei under a stereomicroscope. H. Lactifluus uyedae (AV 12-070). Bars: 2B–E, H = ~10 mm; 2F–G = ~1 mm.
6DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
Figure 3. Microscopic features of Lf. auriculiformis (holotype AV 12-050). A. Basidiospores. B. Basidia. C. Marginal cells. D. Terminal
elements of the pileipellis. E. Pleuropseudocystidia. Bars = 10 µm.
1142 m. Growing on naked soil, on a vertically exposed site
underneath Lithocarpus in rainforest dominated by
Castanopsis and Lithocarpus polystachyus, 16 Jul 2012, A.
Verbeken AV 12-050 (holotype GENT, isotype MFLU).
GenBank: ITS = KR364086; 28S = KR364216.
Etymology: auriculiformis (Latin), with the shape of
an ear, referring to the ear-shaped or pleurotoid
Diagnosis: Basidiocarps small, pure white, and pleur-
otoid, with long and thick-walled pileipellis hairs visible
with a hand lens. Latex white but staining lamellae
brown. Pileipellis a lampropalisade or hymeniderm
with scattered thick-walled terminal elements.
Macrocystidia very abundant and with an acicular,
needle-like content. Basidiospores subglobose to
broadly ellipsoid, with ornamentation forming a
Figure 4. Additional microscopic features of Lf. auriculiformis (holotype AV 12-050). A. Pleuromacrocystidia. B. Pileipellis. Bars = 10 µm.
8DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
subcomplete reticulum with small meshes. Differs from
Lf. conchatulus by abundant and emergent pleuropseu-
docystidia, slightly larger macrocystidia, larger and
more scattered pileipellis hairs, larger spores with a
denser reticulum with distinctly smaller and more irre-
Description: Basidiocarps pleurotoid. Pileus ear- or
shell-shaped,atmost7–10 mm diam but often smal-
ler, pure white, smooth, transparently striate. Stipe
completely eccentric and very short (1–3mm),
slightly more cream-colored than the pileus.
Lamellae white, staining brownish to grayish brown
by the latex, in some specimens remarkably few,
about 5–7 lamellae per pileus, generally a bit more,
with lamellulae. Context white, taste mild. Latex
scarce but visible, watery white, staining the lamellae
cream to distinctly brownish-grayish after more than
Basidiospores subglobose to broadly ellipsoid, (7.4)
7.5–8.3–8.9 × 6.2–6.8–7.3 µm (Q = 1.11–1.22–1.31, n =
30); ornamentation amyloid, composed of rather thick
and irregular, rounded ridges, up to 0.6(0.8) µm high,
ridges forming a dense and subcomplete reticulum with
rather small meshes; plage slightly distally amyloid.
Basidia 60–80 × 13–18 µm, cylindrical to narrowly
clavate, 4-spored, sterigmata 5–11 × 2–5 µm.
Pleuromacrocystidia very abundant, very emergent,
70–120 × 12–14(20) µm, cylindrical and sometimes
with rounded apex, or with very small papilla, or fusi-
form with tapering apex, with slightly refringent walls
and distinct acicular, needle-like content.
Pleuropseudocystidia abundant, slightly emergent, 3–6
µm diam, slightly tortuous, sometimes branched.
Lamella edge substerile, composed of marginal cells
and abundant cheilopseudocystidia, with some cheilo-
macrocystidia and sometimes with small basidia; mar-
ginal cells shortly cylindrical to subclavate, 15–28(35) ×
7–10 µm; cheilomacrocystidia scarce, clavate, 50–70 ×
12–15 µm, with distinct acicular, needle-like content.
Hymenophoral trama composed of hyphae mixed with
lactiferous hyphae. Pileipellis a lampropalisade to
hymeniderm consisting of a layer of rounded cells up
to 50 µm thick, with some bearing rounded to subcla-
vate terminal cells, but also with long, hair-shaped,
thick-walled elements that sometimes arise deep in
the subpellis; hair-like terminal elements 65–190 ×
6–12 µm, broader at the base, becoming narrower at
the apex, with very thick walls (up to 3 µm thick).
Clamp connections absent.
Ecology and distribution: On naked soil on a verti-
cally exposed site underneath Lithocarpus in rainforest
dominated by Castanopsis and Lithocarpus polysta-
Notes:ThepileusofLf. auriculiformis is smooth, but
with a good hand lens some hairs (as thick-walled term-
inal elements in the pileipellis) are visible. The possible
sister species Lf. conchatulus (FIG. 1), described from
similar habitats in Thailand, differs by smaller spores
(6.1–7.8 × 5.1–6.6 µm) and somewhat smaller macrocys-
tidia. However, macrocystidia are mentioned to be only
40–65 µm long in the description of the type, but we did
observe macrocystidia up to 80–90 µm long in Lf. con-
chatulus. Besides the larger macrocystidia, Lf. auriculifor-
mis has larger and more scattered pileipellis hairs. They
measure 65–190 × 6–12 µm compared with 20–70 × 3–6
µm in Lf. conchatulus. The ornamentation of the spores in
Lf. conchatulus is a more regular reticulum with wider
meshes and regular ridges that have a more equal height.
In Lf. auriculiformis, the reticulum is denser with dis-
tinctly smaller meshes that are more irregular as the
height of the ridges is unequal. The reticulum is incom-
plete with numerous open ends. Pleuropseudocystidia are
abundant and emergent in Lf. auriculiformis but scarce
and not emergent in Lf. conchatulus. The margin of the
lamellae is substerile in both species, with some scarce
basidia present, but mainly composed of marginal cells. In
Lf. auriculiformis, cheilopseudocystidia are abundant,
cheilomacrocystidia are scarce, whereas in Lf. conchatulus
cheilomacrocystidia are abundant and cheilopseudocysti-
dia hardly observed.
The spore ornamentation of Lf. auriculiformis is
most similar to the ornamentation in Lf. uyedae, but
this species differs by shorter pileipellis hairs (25–70 ×
2–6μm). When we compare Lf. auriculiformis with Lf.
genevievae, the latter has spores that are comparable in
size (7.1–9.6 × 6.5–8.2) but has a complete and regular
reticulum with larger meshes, similar to Lf. conchatulus.
Furthermore, macrocystidia in Lf. genevievae are from a
completely different type: with a more granular and
dense content instead of distinct acicular, needle-like
content, and with a fusiform shape very gradually
tapering towards the apex.
Lactifluus bhandaryi Verbeken & De Crop, sp.
nov. FIG. 5
Typification: NEPAL. BAGMATI: Kathmandu,
Shivarpuri Watershed Management Area, Malpokhari,
27°47′30″N, 085°22′40″E, 7 Jul 1990, H.R. Bhandary &
Sunar HRB 74 (holotype TENN 051830). GenBank: ITS
= KR364111; 28S = KR364140.
Etymology: bhandaryi (Latin), referring to H. R.
Bhandary, who first collected and documented the spe-
cies (Bhandary 1993).
Diagnosis: Basidiocarps small and pleurotoid, cream-
to orange-colored covered with small hairs, especially
Figure 5. Microscopic features of Lf. bhandaryi (holotype TENN 051830). A. Basidia. B. Pleuromacrocystidia. C. Basidiospores. D.
Pleuropseudocystidia. E. Pileipellis. Bars = 10 μm.
10 DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
when young, and with an acrid taste. Growing on a
white silky subiculum with erect hairs. Pileipellis a
lampropalisade. Pleuromacrocystidia emergent and
long. Basidiospores subglobose to broadly ellipsoid
ornamented by a complete reticulum. Most closely
related to Lf. raspei (described below) but differs from
it by cream to orange basidiocarps in mature stage, long
and very emergent pleuromacrocystidia, and pileipellis
hairs with rounded apex. Differs from Lf. uyedae by
cream to orange basidiocarps in mature stage, unchan-
ging latex, acrid taste, and long and emergent
Description: Basidiocarps pleurotoid, growing on a
subiculum. Subiculum thinly or densely effused around
the substratum, extending up to 60 mm from the basi-
diocarps; white, silky, with erect hairs; hairs scattered,
erect, spiny, fasciculate, sometimes bifurcate with
pointed and curved tips. Pileus flabelliform or spathu-
late, with convex but later slightly depressed at the
center, up to 9 × 12 mm, broadly striate, sulcate, pure
white when young, then white only between the furrow
of striations; cream (4A3), pale yellow (4A4), putty
(4B2), ivory (4B3), champagne (4B4), pale blonde to
blonde (4C3–4) to pale orange (5AB3) or grayish
orange (6D4) or grayish brown (6E4–5) all over or
mostly at the ridges and in the center; surface densely
hairy when young, remaining so towards margin and
pruinose or woolly in the center; margin striate, sulcate,
incurved at first, then straight, slightly lobed (some-
times rimulose and strongly lobed), with erect and
silky hairs all over. Stipe sometimes absent, when pre-
sent cylindrical, strongly eccentric or lateral, up to
1.5 mm long and 0.9 mm diam; surface white, some-
times with hairs up to 1 mm long, base white and
covered with a strigose hairy tomentum. Lamellae
decurrent, white to yellowish white (4A2), up to
1.5 mm deep, distant, furcate up to one or two levels,
with lamellulae. Context very thin, up to 0.3 mm in the
center of the pileus, white or grayish white, turning
pink or pinkish brown with age, brittle. Latex abun-
dant, white, unchanging. Taste immediately acrid. Odor
Basidiospores subglobose to broadly ellipsoid, 6.8–
7.7–8.5 × 6.0–6.6–7.3 µm (Q = 1.11–1.1–1.21, n = 20);
ornamentation amyloid, dense, composed of ridges and
warts up to 0.7(–1) µm high, usually connected by rather
thick and high ridges, forming a complete reticulum;
plage sometimes totally amyloid. Basidia 40–50 × 9–11
µm, subclavate, 4-spored. Pleuromacrocystidia abun-
dant, 90–165 × 12–15 µm, strongly emergent, fusiform,
tapering near the apex, usually thin-walled, sometimes
clearly partially thick-walled, with dense acicular, nee-
dle-like content. Pleuropseudocystidia not abundant,
2–4 µm diam, cylindrical and narrow, not emergent.
Lamella edge sterile, with marginal cells and cheiloma-
crocystidia; marginal cells shortly cylindrical to subcla-
vate, 17–27 × 4–7 µm; cheilomacrocystidia similar to
pleuromacrocystidia. Pileipellis a lampropalisade; sub-
pellis up to 30 µm thick, consisting of globose to sub-
globose cells, 15–25 µm diam; suprapellis with long,
hair-shaped, thick-walled, often septate elements,
40–100 × 7–9 µm, sometimes distinctly broader at the
base (up to 15 µm), becoming narrower at the top but
apex rounded, never acute. Clamp connections absent.
Ecology and distribution: Gregarious on roots of
Castanopsis tribuloides, Myrsine semiserrata, and M.
capitellata but overgrowing on other closely associated
leaf litter, soil, decaying sticks, stem base, and roots of
ferns and other angiosperms such as Daphne bholua,
Myrica esculenta, Quercus leucotrophora, and Q. lamel-
Other specimens examined: NEPAL. BAGMATI:
Kathmandu, Nagajun (Raniban) forest, 27°45′00″N,
085°15′25″E, 10 Jul 1991, H.R. Bhandary HRB 58
(TENN 051829); ibidem, 27°47′30″N, 085°22′40″E, 9
Jul 1990, H.R. Bhandary HBR 135 (TENN 051831);
ibidem, 27°45′00″N, 085°15′25″E, 15 Jul 1991, H.R.
Bhandary HRB 83 (TENN 051832).
Notes: Lactifluus bhandaryi is most closely related to
Lf. raspei and Lf. uyedae. Macroscopically, it differs
from both species by its cream to orange basidiocarps
in mature stage, whereas both other species have pure
white basidiocarps. Furthermore, the latex of Lf. uyedae
stains the lamellae brownish after 30 min, whereas the
latex of Lf. bhandaryi is unchanging, and Lf. bhandaryi
has an acrid taste but the taste of Lf. uyedae is mild.
Although the fruit bodies are very small, the latex is
abundant enough to taste the latex separately from the
flesh. Microscopically, Lf. bhandaryi has long and very
emergent pleuromacrocystidia (90–165 × 12–15 µm),
whereas pleuromacrocystidia of Lf. raspei are much
shorter (50–60 × 7–12 µm) and those of Lf. uyedae
are shorter (75–95(120) × 8–11 µm), not very emergent
but arising very deep in the subhymenium. The pilei-
pellis hairs of Lactifluus bhandaryi have rounded
apices, whereas those of Lf. raspei can be very acute.
Lactifluus gerardiellus Wisitrassameewong &
Verbeken, sp. nov. FIGS. 2C,6
Typification: THAILAND. CHIANG RAI
PROVINCE: Thasai Sub-district, Muang District, Doi
Pui, television repeater station, 19°49′00″N, 99°52′03″E,
740 m. Gregarious among leaf litter in deciduous forest
dominated by Castanopsis armata and Quercus, 31 Jul
2012, K. Wisitrassameewong, J. Chen & B. Thongbai
Figure 6. Microscopic features of Lf. gerardiellus (holotype KW 386). A. Cheilomacrocystidia. B. Basidia. C. Basidiospores. D. Pileipellis.
Bars = 10 μm.
12 DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
KW 386 (holotype GENT, isotype MFLU). GenBank:
ITS = KX889845; 28S = KX889844.
Etymology: gerardiellus (Latin), resembling a small
version of Lactifluus gerardii.
Diagnosis: Basidiocarps small and agaricoid, resem-
bling small forms of Lf. gerardii. Pileus and stipe brown
with white lamellae. Pileipellis a lampropalisade with
thick-walled, hair-like elements. Basidiospores subglo-
bose to ellipsoid, with ornamentation forming an
incomplete reticulum. Forming a sister lineage to Lf.
indicus, from which it differs by an agaricoid habit, the
absence of subiculum, longer pileipellis hairs, longer
basidia, and lower spore ornamentation.
diam, applanate to infundibuliform, more or less mucro-
nate in the center, margin striate; edge even; surface dry,
velvety, even to slightly wrinkled; sometimes with uneven
color ranging from dark brown to reddish brown (7E5,
7F6–7, 8E7–8). Stipe 5–8mmlong,1–2mmdiam,cylind-
rical to slightly eccentric, color often paler at apex (5A3,
5B3–B4), white pruinose at base, with hollow pith.
Lamellae close, white, with two series of lamellulae.
Context <0.5 mm thick in the pileus, cream to pale yellow,
not discoloring when bruised, FeSO
mild, odor sweetish. Latex rather sparse, white or watery
white, unchanging when isolated on glass slide, 10% KOH
on latex yellow (4C6); taste mild. Spore print white.
Basidiospores subglobose to ellipsoid, mostly broadly
ellipsoid, 6.7–7.6–8.4 × 5.4–6.1–6.8(6.9) µm (Q = 1.11–
1.25–1.39, n = 30); ornamentation amyloid, composed of
ridges and interconnected warts up to 0.5 µm high, form-
ing an incomplete reticulum; plage slightly distally amyloid.
Basidia 65–75 × 11–14 µm, cylindrical to subcylindrical, 4-
spored. Pleuromacrocystidia absent. Pleuropseudocystidia
3–5 µm diam, slightly tortuous. Lamella edge sterile, com-
posed of marginal cell and abundant cheilomacrocystidia;
marginal cells shortly cylindrical to subclavate, often nar-
row and tortuose, 10–20 × 4–8 µm; cheilomacrocystidia
55–84 × 8–12 µm, fusiform. Hymenophoral trama com-
a lampropalisade; subpellis consisting of globose to sub-
globose cells, 15–30 µm diam, forming a dense layer up to
60 µm thick; suprapellis with long, hair-shaped, thick-
walled elements, 25–90 × 6–10 µm, broader at the base,
becoming narrower at the apex, with dark intracellular
pigmentation. Clamp connections absent.
Ecology and distribution: Gregarious among leaf lit-
ter in deciduous forest dominated by Castanopsis
armata and Quercus. Thailand.
Notes: Lactifluus gerardiellus forms a sister lineage to Lf.
indicus. Macroscopically, they differ in habit, as Lf. gerar-
diellus is a small agaricoid species whereas Lf. indicus is a
pleurotoid species that grows on a subiculum (Latha et al.
2016). Microscopically, they differ because Lf. indicus has
pileipellis hairs that are shorter (9–48 × 4–7μm) and more
rounded at the apex, shorter basidia (24–51 × 7–13 μm),
and a higher spore ornamentation (up to 2.7 µm).
Lactifluus gerardiellus is macroscopically similar to Lf. par-
vigerardii, with paler and more yellowish brown cap colors.
Microscopically, Lf. parvigerardii hassporeswithahigher
ornamentation and a centrally amyloid plage and the
terminal elements of the pileipellis are thin-walled and
smaller than in Lf. gerardiellus. Lactifluus parvigerardii
has pleuromacrocystidia with a more or less rounded
apex and no cheilomacrocystidia, whereas Lf. gerardiellus
has only cheilomacrocystidia with a pointed apex.
Moreover, in the type collection of Lf. gerardiellus,consid-
erably larger basidia have been observed.
Lactifluus pulchrellus F. Hampe & Wisitrassameewong,
sp. nov. FIGS. 2D,7,8; SUPPLEMENTARY FIG. 1B
Typification: THAILAND. CHIANG MAI
PROVINCE: Mae Teang District, Buatong Waterfall
and Rainbow Spring, 19°04′11.78″N, 99°04′48.41″E,
507 m, on naked stony soil under Dipterocarpus tuber-
culatus, 23 Jun 2012, F. Hampe & K.
Wisitrassameewong KW 304/FH 12-037 (holotype
GENT, isotype MFLU). GenBank: ITS = KR364092;
28S = KR364223.
Etymology: pulchrellus (Latin), refers to the beauti-
fully small basidiocarps of this species.
Diagnosis: Basidiocarps small and agaricoid. Both
pileus and stipe velutinous and bright orange-red.
Lamellae rather thick, cream- to yellow-colored and
staining brownish black by the latex. Latex watery
greenish brown, staining lamellae and context brownish
black when bruised. Pileipellis a lamprotrichopalisade.
Basidiospores subglobose to broadly ellipsoid, with an
ornamentation of irregular warts sometimes connected
by fine lines. Differs from Lf. igniculus by basidiospore
ornamentation of low and irregular rounded warts and
from Lf. parvigerardii by orange to red basidiocarp
colours, lamprotrichopalisade as pileipellis, and lower
and less connected basidiospore ornamentation.
Description: Basidiocarps small, fragile. Pileus
2–11.5 mm in diam, convex at first, then applanate,
occasionally lobate; center at first sometimes papillate,
later depressed; margin at first regular and bent down-
wards, later wavy; yellowish orange, bright orange, to
orange reddish (5B7, 6C7–8, 6B8, 7D8), more intensive
orange-red (8C–D8, 9C–D8) when young, often par-
tially paler at maturity (6D6); surface velvety, dry,
rimose and rivulose, sometimes with concentric cracks,
at times striate almost up to the center. Stipe very small,
3–5 × 0.5–1 mm, equal, cylindrical or thickening
towards base, velutinous, concolourous with the cap or
somewhat more pinkish, base more pale, sometimes
with fine white tomentum, immediately greenish blue
with guaiac. Lamellae adnate, about 10–20 per pileus,
moderately spaced with 1–3 lamellulae, relatively thick
(1 mm); cream to yellow, staining brownish to brownish
black (2F1) by the latex. Context 0.3–0.5 mm thick in the
pileus, cream to pale yellow, not discoloring where
Figure 7. Microscopic features of Lf. pulchrellus (holotype KW 304/FH 12-037). A. Basidia. B. Pileipellis. Bars = 10 μm.
14 DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
Figure 8. Additional microscopic features of Lf. pulchrellus (holotype KW 304/FH 12-037). A. Marginal cells. B. Basidiospores. C.
Pleuromacrocystidia. D. Terminal elements of the pileipellis. Bars = 10 μm.
bruised; taste mild. Latex abundant, watery greenish
brown (2F3–5), staining lamellae and flesh brownish
cream at first, then blackish (2F1), 30% KOH on latex
yellow (4C7); taste mild. Spore print white.
Basidiospores subglobose to broadly ellipsoid, 6.9–
7.5–8.1 × 5.9–6.5–7.1 µm (Q = 1.07–1.17–1.28, n = 30);
ornamentation amyloid, with irregular warts up to 0.3
µm high that are sometimes connected by fine lines,
sometimes isolated; plage distally or almost completely
and distinctly amyloid. Basidia 55–70 × 11–15 µm, 4-
spored, sometimes 2-spored and then often with very
long and irregular sterigmata. Pleuromacrocystidia
70–95 × 10–15 µm, cylindrical to subfusiform, often
with rounded to slightly tapering apex, sometimes with
long tapering apex, thin-walled. Pleuropseudocystidia
2–4 µm diam, cylindrical. Lamella edge sterile; cheilo-
cystidia absent; marginal cells cylindrical to subclavate,
thin-walled, and hyaline, 15–50 × 7–15 µm.
Hymenophoral trama mixed, composed of hyphae
and some small sphaerocytes. Pileipellis a lamprotri-
chopalisade with chains of short, irregular, or subglo-
bose elements, with the terminal element of the chain
clavate to globose and sometimes slightly thick-walled,
mixed with long and rather slender distinctly thick-
walled hairs; hair-like terminal elements up to 140 µm
long, 12–14 µm diam, very thick-walled, often septate,
usually tapering upwards. Clamp connections absent.
Ecology and distribution: On naked stony soil under
Dipterocarpus tuberculatus. Thailand.
Notes: This is a striking species due to the small
dimensions of the basidiocarps and the warm red to
orange or even somewhat pinkish colors. The species
stands out in Lactifluus sect. Gerardii, which has mainly
dark brown to blackish brown or very pale to whitish
representatives. Recently, Morozova et al. (2013)
described a species in this section with deep orange
tinges—Lf. igniculus O.V. Morozova & E.S. Popov.
They describe the species as pleurotoid, but the pictures
in their description show agaricoid basidiocarps with a
central to slightly eccentric stipe, which is macroscopi-
cally very similar to the proposed species here.
However, the spore ornamentation differs considerably
between the two species (FIG. 8): Lf. pulchrellus has low
and irregular rounded warts, whereas Lf. igniculus has
almost echinulate spores with acute warts up to 1.6 µm
high (our own measurements), isolated or connected by
fine lines. Morozova et al. (2013) also described a
collection of Lf. aff. igniculus that is close to, but not
conspecific with, Lf. igniculus. The basidiocarps look
strikingly similar, and in the molecular analysis their
ITS sequences only differ by a few base pairs from those
of both Lf. igniculus and Lf. pulchrellus. Nonetheless, Lf.
aff. igniculus differs morphologically from Lf. pulchrel-
lus, with echinulate spores with warts up to 1 µm.
Lactifluus raspei Verbeken & De Crop, sp. nov.
FIGS. 2E–G,9,10; SUPPLEMENTARY FIG. 1C–D
Typification: THAILAND. CHIANG MAI
PROVINCE: Mae Taeng District, Baan Mae Sae, 19°
14′26″N, 98°37′60″E, 1077 m, on soil and seedlings in
mixed forest of Dipterocarpus, Castanopsis, Lithocarpus,
and Quercus, 1 Aug 2014, O. Raspé EDC 14-517 (holo-
type GENT, isotype MFLU). GenBank: ITS =
Etymology: raspei (Latin), named after Dr. Olivier
Raspé, who found and collected the species.
Diagnosis: Basidiocarps small, white, and pleurotoid,
forming a white subiculum on soil or plant seedlings.
Basidiocarps covered with white hairs visible to the
naked eye. Latex white and unchanging. Pileipellis a
lampropalisade. Basidiospores subglobose to broadly
ellipsoid, ornamentation forming a subcomplete reticu-
lum. Differs from Lf. bhandaryi by its white basidio-
carps, shorter pleuromacrocystidia, and pileipellis hairs
with acute apices. Differs from Lf. uyedae by the pre-
sence of subiculum, its velvety surface, and shorter
Description: Basidiocarps small, pleurotoid, growing
on a subiculum. Subiculum white to grayish white,
thinly to moderately densely effused when growing on
soil, densely effused on plant seedlings. Pileus 4–7mm
diam, plano-convex when young, applanate when
older; young basidiocarps sometimes papillate,
depressed when older; margin slightly inflexed; margin
edge entire; white, with yellow tinge when old; surface
velvety, covered with hairs. Stipe very small, 2–4 × 0.5–
1 mm, laterally attached, cylindric, tapered towards the
base, velutinous, with a white and hairy tomentum at
the base, concolourous with the pileus. Lamellae nar-
rowly adnate, about 9–12 lamellae per pileus with 1–5
lamellulae, rather distant (in proportion to its size),
relatively thick, white, edges concolorous and entire.
Context 0.3–0.5 mm thick in the pileus, white, no
color change where bruised. Latex not abundant,
white, no color change. Spore print white.
Basidiospores subglobose to broadly ellipsoid, (6.8)
6.9–7.8–8.8(9.0) × 5.7–6.7–7.6(8.0) µm (Q = 1.09–1.18–
1.27, n = 20); ornamentation amyloid, dense, composed
of interconnected warts up to 1 µm high, usually con-
nected by lower ridges, forming a subcomplete reticu-
lum; plage often totally amyloid. Basidia 40–45 × 10–11
µm, subcylindrical to subclavate, 4-spored.
Pleuromacrocystidia abundant, 50–60 × 7–12 µm,
16 DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
emergent, irregularly subclavate, sometimes narrower
near apex, usually thin-walled, sometimes slightly and
locally thick-walled, with a dense acicular, needle-like
content. Pleuropseudocystidia very abundant, 2–3µm
diam, cylindrical and very narrow, slightly tortuous, not
emergent to emergent. Lamella edge mixed, with basi-
dia, pseudocystidia, and marginal cells; marginal cells
shortly cylindrical to subclavate, 10–25 × 4–8 µm; basi-
dia distinctly smaller than the basidia at the face of the
lamellae, 25–30 × 7–8 µm. Pileipellis a lampropalisade;
subpellis up to 40 µm thick, consisting of globose to
subglobose cells, 10–15 µm diam; suprapellis with long,
hair-shaped, thick-walled, and often septate elements,
40–120 × 7–10 µm, broader at the base, becoming
narrower and sometimes very acute at the apex.
Subiculum composed of hyaline hyphae which are 2–7
µm wide, septate, thick-walled, walls ±1 µm wide.
Clamp connections absent.
Ecology and distribution: On soil and seedlings in
mixed forest with Dipterocarpus, Castanopsis,
Lithocarpus, and Quercus. Thailand.
Notes: Lactifluus raspei is most closely related to Lf.
bhandaryi and Lf. uyedae. Lactifluus bhandaryi differs
macroscopically by its cream to orange basidiocarps in
mature stage and by its unchanging latex.
Microscopically, it has longer and more emergent
pleuromacrocystidia (90–165 × 12–15 µm) and pilei-
pellis hairs with rounded apices. Lactifluus uyedae
Figure 9. Microscopic features of Lf. raspei (holotype EDC 14-517). Pileipellis. Bar = 10 μm.
Figure 10. Additional microscopic features of Lf. raspei (holotype EDC 14-517). A. Marginal cells. B. Elements of the lamella edge. C.
Basidia. D. Pleuropseudocystidia. E. Pleuromacrocystidia. F. Basidiospores. Bars = 10 μm.
18 DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
differs macroscopically from Lf. raspei by its smooth
surface, latex that stains the lamellae brownish after 30
min, and the absence of subiculum. Microscopically, it
has longer pleuromacrocystidia (75–95(120) × 8–11
µm) that are not very emergent but arising very deep
in the subhymenium.
Lactifluus uyedae (Singer) Verbeken, Mycotaxon
119:485. 2012.FIGS. 2H,11,12
Basionym: Lactarius uyedae Singer, Nova Hedwigia
Description: Basidiocarps small, pleurotoid. Pileus
ear-shaped, shell-shaped, up to 7–10 mm diam,
often smaller, pure white, smooth, transparently
striate. Stipe completely eccentric and very short
(1–3 mm), slightly more cream-colored than the
pileus. Lamellae white, then staining brownish to
grayish brown by the latex, in some specimens
with remarkably few lamellae (about 5–7perpileus),
usually a bit more, lamellulae also present. Context
white; taste mild. Latex scarce but visible, watery
white, staining the lamellae cream at first but after
more than 30 min becoming distinctly brownish
Basidiospores subglobose to broadly ellipsoid, (7.5)
7.6–8.3–9.1 × 6.3–7.1–7.8 µm (Q = 1.09–1.18–1.26), n =
20; ornamentation amyloid, composed of rather thick
and irregular, rounded ridges, up to 0.8 µm high, form-
ing a dense, subcomplete reticulum with rather small
meshes; plage slightly distally amyloid. Basidia 55–65
(75) × 9–15 µm, mostly cylindrical, sometimes nar-
rowly clavate, 4-spored, rarely 2-spored; sterigmata
5–8×1–3 µm. Basidioles distinctly and strikingly mul-
tiseptate. Pleuromacrocystidia very abundant, usually
not very emergent but arising very deep in the subhy-
menium, 75–95(120) × 8–11 µm, clavate to fusiform
with rounded or rather abruptly tapering apex, with
distinct acicular, needle-like content.
Pleuropseudocystidia abundant, not to slightly emer-
gent, usually very narrow, 2–4(5) µm diam, slightly
tortuous. Lamella edge substerile, composed of mar-
ginal cells and abundant cheilomacrocystidia, only a
few basidia present; marginal cells shortly cylindrical
to subclavate, multiseptate, 28–50 × 7–12 µm; cheilo-
macrocystidia fusiform to irregularly fusiform, 55–75 ×
10–15 µm. Hymenophoral trama composed of hyphae,
mixed with lactiferous hyphae. Pileipellis a palisade to
hymeniderm, consisting of a layer of rounded cells up
to 60 µm thick, with some bearing either rounded to
subclavate terminal cells or long, hair-shaped, thick-
walled elements; hair-like terminal elements 90–180 ×
6–12 µm, septate, sometimes swollen at the base,
becoming narrower at the apex, very thick-walled.
Clamp connections absent.
Other specimens examined: JAPAN. SHIGA: Otsu,
Kokubu, Aug 1973 to Sep 1974, Uyeda s.n. (holotype
F). THAILAND. CHIANG MAI PROVINCE: Mae
Teng District, Ban Pa Deng, Pathummikaram Temple,
19°06.77′N, 98°44.32′E, 1030 m, on naked soil under
Lithocarpus, Shorea, and Castanopsis, 18 Jul 2012, A.
Verbeken AV 12-070 (GENT, MFLU).
Notes: The Thai collection (AV 12-070) is closely
related to Lf. uyedae. Molecular analyses show a very
close relationship with the sequence from type material
of Lf. uyedae (99% sequence identity). This result is
only based on 28S sequences, as only 28S is available
of the type specimen. Basidiospore size and ornamen-
tation of the Thai collection are comparable to those of
the type collection (Verbeken 1998). The macrocystidia
that we measured in the Thai specimen are comparable
to those of the type specimen as well (AV 12-070:
75–95 × 8–11 µm; type: 90–100(120) × 7–9 µm). We
did observe differences in the terminal hairs in the
pileipellis. In the Thai collection, these hairs are rather
long (90–180 × 6.3–7.8 µm), whereas those of the type
collection are shorter (20–80 × 6–8 µm). More speci-
mens are needed in order to find out whether this is
indeed an informative feature at species level. A strik-
ing feature of the Thai collection is the presence of
abundant multiseptate basidioles in the hymenium.
Often the upper cell is very short, and it is not clear
whether they actually develop into true and mature
Milkcaps with pleurotoid or small agaricoid basidio-
carps are rare, and it is only occasionally that new
collections are found. Moreover, they often have tiny
basidiocarps that are easily overlooked in the field.
They are only known from the tropics and long
believed to have isolated positions in the Lactifluus
phylogeny. Our new findings indicate that pleurotoid
diversity of Lactifluus in Asia is greater than previously
We only have single collections of four of these new
species and acknowledge that this is not favorable for
describing new species. However, we prefer to describe
them as new for the following reasons. First, it is
unlikely that multiple new collections of all these spe-
cies will be found soon. Four of the new species we
describe in this paper were found near the Mushroom
Research Centre in Chiang Mai, Thailand. These local-
ities were frequently visited by members of our labora-
tory and others during the past decade, but only a
handful of expeditions encountered pleurotoid collec-
tions. Second, all newly described species do show
Figure 11. Microscopic features of Lf. uyedae (AV 12-070). A. Basidiospores. B. Cheilomacrocystidia. C. Basidia. D. Marginal cells. E.
Pleuromacrocystidia. F. Pleuropseudocystidia. Bars = 10 μm.
20 DE CROP ET AL.: NEW PLEUROTOID AND SMALL AGARICOID LACTIFLUUS SPECIES
unique morphological and molecular characteristics,
which makes it possible to describe and delineate
them from closely related species.
With the description of five new species from
Lactifluus sect. Gerardii, we confirm once more that
this section contains a large and partially unknown
diversity. Stubbe et al. (2010) already demonstrated
the molecular and morphological diversity of this
section, which mainly contains species with brownish
or white basidiocarps. The findings of brightly
orange-red-colored species, such as Lf. pulchrellus
and the species found by Morozova et al. (2013),
emend the characters of the group, which was
thought to be characterized by either white, pleuro-
toid species or agaricoid species with dark brown to
blackish pigments. Taking the results of this study
into account, Lf.sect.Gerardii now contains 20
described Asian species.
When Stubbe et al. (2010) published the first com-
prehensive account on Lactifluus sect. Gerardii based
on worldwide sampling with a combined molecular and
morphological approach, they were able to distinguish
three major clades within the section: (i) the/gerardii
clade with the typical representatives of the section
characterized by the lack of macrocystidia; (ii) the/
uyedae clade containing species with very small, pale-
colored, pleurotoid basidiocarps; and (iii) the/ochroga-
lactus-petersenii clade characterized by basidiomes with
a discoloring latex. This concept was challenged by the
discovery of Lf. parvigerardii, a taxon with very small,
dark-colored, agaricoid basidiocarps with discoloring
latex, and presence of macrocystidia. Molecular results
showed that this species is closely related to the/uyedae
clade (Wang et al. 2012), which was not expected based
on morphology alone. The new taxa presented by
Morozova et al. (2013) and Latha et al. (2016), together
Figure 12. Pileipellis of Lf. uyedae (AV 12-070). Bar = 10 μm.
with those from the present paper, contribute to a new
understanding of the subdivisions within Lactifluus
sect. Gerardii. On this basis, two more clades are recog-
nized within the section: (iv) the/parvigerardii clade,
which contains taxa with tiny agaricoid basidiocarps
and is characterized microscopically by the lack of
cheilomacrocystidia. In this lineage, the strikingly
vivid basidiocarp colors presented by the Vietnamese
Lf. igniculus (Morozova et al. 2013) and the newly
described Thai Lf. pulchrellus are a unique feature
within the whole section; and (v) the/uyedae clade,
which in Stubbe et al. (2010) exclusively contained
whitish pleurotoid taxa but is now joined by agaricoid
and pleurotoid representatives with more typical colors
of the section (Lf. gerardiellus and Lf. indicus) and a
pleurotoid species with cream- to yellowish-orange-
colored basidiocarps (Lf. bhandaryi). These findings
extend the morphological circumscription of this line-
age. On this basis, the/uyedae clade contains taxa with
very small basidiocarps characterized by the presence of
macrocheilocystidia and macropleurocystidia.
Our results confirm that species with pleurotoid
basidiocarps and small basidiocarps originated multiple
times within the genus Lactifluus. Within the genus,
species with pleurotoid basidiocarps originated at least
five times, and species with small basidiocarps at least
six times. The transition from medium to large agar-
icoid basidiocarps to small agaricoid basidiocarps and/
or small pleurotoid basidiocarps thus occurs multiple
times, suggesting that it might be evolutionary advan-
tageous and rather easy. Reversals from pleurotoid or
small agaricoid basidiocarps have not been recorded
yet. From our data we cannot decide whether there is
a relationship between the small agaricoid habit and the
pleurotoid habit. Only in theuyedae clade there seems
to be a transition from species with small agaricoid
basidiocarps to pleurotoid basidiocarps. In all other
clades, such a relationship is not obvious.
Many pleurotoid species were found on the bark of
trees, on seedlings, on roots, or on naked soil that is
vertically exposed. This might indicate that the pleur-
otoid habit has some advantages in this habitat type as
opposed to agaricoid basidiocarps. As Henkel et al.
(2000) wrote in their study of pleurotoid Russulaceae
of Guyana and Japan, it remains unclear what the
reason is for an elevated position of basidiocarps.
Investing in smaller basidiocarps also seems an advan-
tage in conditions where the fruit body is more suscep-
tible to rotting or damage before the spores are even
dispersed. We hypothesize that Lactarius has a way of
escaping environmental stress by going angiocarpic,
whereas Lactifluus does this by forming different
small basidiocarps to spread the risk.
We thank R. Petersen for providing herbarium collections of
Lactifluus bhandaryi and H. R. Bhandary for providing ela-
borate descriptions of the species. We thank Olivier Raspé for
providing collections of Lactifluus raspei. We thank the
reviewers and the editor for their constructive suggestions
and detailed comments on the manuscript.
E. De Crop and K. Wisitrassameewong are supported by the
“Special Research Fund Ghent University”(BOF: E. De Crop,
grants B/13485/01 and BOF-PDO-2017-001201; K.
Wisitrassameewong, grant B/11696/02).
Eske De Crop http://orcid.org/0000-0002-9067-6981
Felix Hampe http://orcid.org/0000-0001-7642-7930
Komsit Wisitrassameewong http://orcid.org/0000-0003-
Dirk Stubbe http://orcid.org/0000-0002-2502-2180
Jorinde Nuytinck http://orcid.org/0000-0001-9250-377X
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